Staphylococcus aureus is emerging as the most problemafic bacterial pathogen facing our community and healthcare settings. An effective strategy for S. aureus to survive in the host is to attach to a surface and develop into an encased community of cells called a biofilm. We recently discovered that quorum-sensing can control the balance between a planktonic or biofilm lifestyle, suggesting that modulation of this dispersal mechanism could be an effective therapeutic strategy. In collaboration with Dr. Kenneth Bayles (the PI of this PPG), we demonstrated that a deletion of the S. aureus secreted nuclease (Nuc) caused an overall thickening of the biofilm and inhibited secondary structure formation, and we have confirmed a recent report that S. aureus possesses a second extracellular nuclease activity (Nuc2). Based on these findings, our central hypothesis is that control over extracellular nuclease activity is a critical determinant of biofilm maturation and dispersal. To address this question, in Specific Aim 1 we will (i) define the role of Nuc and Nuc2 in biofilm maturation; (ii) determine whether nuclease activity is important for biofilm dispersal; and (iii) modulate biofilm integrity with controlled exposure to nuclease. We further propose that S. aureus nuclease is an important virulence factor. To investigate the nuclease function in disease, we will work with Dr. Tammy Kielian (Project 4 leader) and (i) examine the role of nuclease activity in evasion of neutrophil extracellular traps (NETs); (ii) define the significance of nuclease in mouse models of planktonic versus biofilm infection; and (iii) compare the host inflammatory response to nuclease in planktonic versus biofilm infection. Finally, we speculate that small-molecule inducers of nuclease activity could serve as anti-biofilm therapeutics. Towards this end, in Specific Aim 3, we will employ new technology to generate cyclic peptide libraries in S. aureus that are amenable to high-throughput screening methods. More specifically, we will (i) screen for cyclic peptides that induce nuclease expression through FACS; (ii) perform molecular and biochemical studies to identify peptide targets; (iii) characterize the best candidates as dispersal agents in a biofilm infection model; and (iv) compare results to transposon mutants with increased nuclease activity. Overall, the goal of this Project is to understand how these S. aureus biofilm structures form and disassemble, the contribution of extracellular DNA to this process, and the relevance in disease.